Radiol Oncol 2005; 39(3): 197-210. review Molecular biology of the lung cancer Sasho Z. Panov Laboratory for Molecular Biology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, Skopje, Republic of Macedonia Background. Lung cancer is one of the most common malignant diseases and leading cause of cancer death worldwide. The advances in molecular biology and genetics, including the modern microarray technology and rapid sequencing techniques, have enabled a remarkable progress into elucidating the lung cancer ethiopathogenesis. Numerous studies suggest that more than 20 different genetic and epigenetic alterations are accumulating during the pathogenesis of clinically evident pulmonary cancers as a clonal, multistep process. Thus far, the most investigated alterations are the inactivational mutations and losses of tumour suppressor genes and the overexpression of growth-promoting oncogenes. More recently, the acquired epigenetic inactivation of tu- mour suppressor genes by promoter hypermethylation has been recognized. The early clonal genetic abnor- malities that occur in preneoplastic bronchial epithelium damaged by smoking or other carcinogenes are be- ing identified. The molecular distinctions between small cell lung cancer (SCLC) and non-small cell lung can- cer (NSCLC), as well as between tumors with different clinical outcomes have been described. These inves- tigations lead to the “hallmarks of lung cancer”. Conclusions. It is realistic to expect that the molecular and cell culture-based investigations will lead to discov- eries of new clinical applications with the potential to provide new avenues for early diagnosis, risk assessment, prevention, and most important, new more effective treatment approaches for the lung cancer patients. Key words: lung neoplasms-genetics; genes, tumor suppressor Introduction death worldwide with estimated more than 1.3 million new cases each year.1 The lung cancer Lung cancer is one of the most common ma- incidence and mortality have risen into epi- lignant diseases and leading cause of cancer demic proportions in Western world during the 20th century.2 The majority of lung cancer Received 24 August 2005 patients is inoperable or has disseminated dis- Accepted 11 September 2005 ease at the time of diagnosis and displays a re- Correspondence to: Sasho Z. Panov, PhD, Teaching markable insensitiveness to chemotherapeu- and Research Assistant of Molecular Biology and tics and radiation therapy.3 Over 85% of these Molecular Genetics, Institute of Biology, Faculty of patients eventually die from disseminated dis- Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, Arhimedova bb., MK-1000, ease during the first 5 years and this extreme Skopje, Republic of Macedonia; Phone: 389 70 248 mortality has not changed significantly during 790; Fax: 389 2 3228 141; E-mail: firstname.lastname@example.org the last three decades. Despite diagnostic and 198 Panov SZ / Molecular biology of the lung cancer therapeutic improvements, the 5-year survival of multiple molecular abnormalities over rate has barely increased from 7 to 14% since time. Those alterations lead to acquired cellu- 1970-thies to the present. Moreover, the lung lar capabilities that can be classified in the carcinoma is accounting for nearly 29% of all following six functional sets: a) self-sufficien- cancer-related deaths in both genders, that ex- cy in growth signals due to mutations in pro- ceeds the sum of the next three leading causes to-oncogenes, b) insensitivity to antiprolifera- of death due to breast, colon, and prostate can- tive signals as a result of mutations affecting cer.4 the tumour suppressor genes, c) evading of It is believed that smoking is the primary eti- apoptosis by up-regulation of antiapoptotic or ologic agent in more than 80% of lung cancer down-regulation of proapoptotic molecules, patients.5 The other risk factors include, but are d) limitless replicative potential due to the ac- not limited to, passive smoking, exposure to tivation of telomerase, e) sustained angiogen- environmental pollutants, occupational expo- esis and f) capability for tissue invasion and sure to chemicals (arsenic, asbestos, chromi- capability for dissemination into distant sites um, nickel and vinyl chloride) and to the natu- (metastasis).11 Those molecular alterations ral radioactive gas radon.2 Genetic predisposi- can occur at the level of gene up-regulation or tion, especially polymorphisms of the tumor down-regulation, DNA sequence changes suppressor genes and the allelic variants of the (point mutations), loss of heterozygosity (i.e., genes involved in detoxification, are implicated deletion of one copy of allelic DNA se- into the susceptibility to the disease.6 quences), DNA segment amplification or Based on the histopathological classifica- whole chromosome gains or losses with the tion (WHO, 1977), lung cancer is divided into simultaneous genomic instability and alter- two main types: non–small cell (NSCLC) and ations in microsatellite DNA.12,13 small cell lung cancer (SCLC), which are delin- The advances in molecular biology and ge- eated by their biological and clinical features. netics, including the modern microarray tech- Furthermore, NSCLC consists of several sub- nology and rapid sequencing techniques, types, predominantly adenocarcinoma, squa- have enabled a remarkable progress into elu- mous-cell carcinoma, and large-cell carcinoma. cidating the lung cancer ethiopathogenesis. SCLC is a distinct clinicopathological entity Numerous studies suggest that more than 20 with neuroendocrine pathophysiologic fea- different genetic and epigenetic alterations tures and characteristic microscopic morphol- are accumulating during the pathogenesis of ogy.7 SCLC represents roughly 20% of all pul- clinically evident pulmonary cancers as a monary cancers. The histologic distinction be- clonal, multistep process.14-16 Thus far, the tween NSCLC and SCLC is clinically extreme- most investigated alterations are the inactiva- ly important. There are considerable differ- tional mutations and losses of tumor sup- ences between those two groups in both, ther- pressor genes and overexpression of growth- apeutic approach and prognosis of the disease. promoting oncogenes. More recently, the ac- Recently, molecular classification of lung car- quired epigenetic inactivation of tumor sup- cinomas has been made using mRNA expres- pressor genes by promoter hypermethylation sion profiling by microarray technology.8-10 has been recognized. The early clonal genetic abnormalities that occur in preneoplastic bronchial epithelium damaged by smoking or Molecular biology of lung cancer other carcinogenes are being identified. The molecular distinctions between SCLC and It is generally accepted that the pathogenesis NSCLC, as well as between tumors with dif- of human cancer involves the accumulation ferent clinical outcomes have been described. Radiol Oncol 2005; 39(3): 197-210. Panov SZ / Molecular biology of the lung cancer 199 These investigations lead to the “hallmarks of apoptosis inhibition.20 EGFR is overex- lung cancer”.3 It is realistic to expect that the pressed in the advanced NSCLC, and is asso- molecular and cell culture-based investiga- ciated with the poor survival and resistance tions will lead to discoveries of new clinical to chemotherapeutic agents, including cis- applications with the potential to provide platin. The results of different studies investi- new avenues for early diagnosis, risk assess- gating the prognostic value of EGFR expres- ment, prevention, and most important, new sion in lung cancer are contradictory.3 more effective treatment approaches for the However, since EGFR expression is clearly in- lung cancer patients. volved in the lung cancer pathogenesis, this molecule is an attractive target of different therapeutic approaches.21 Few EGFR in- Growth stimulation by oncogenes hibitors (CP358774, ZD1839-Iressa and OSI774) are under intensive clinical trials in Protein-tyrosine kinases (PTKs) are vital reg- lung cancer patients.3 ulators of intracellular signal-transduction HER-2/neu (ErbB-2) gene is located on pathways that mediate development and cell- chromosome 17p21 and encodes for a 185- to-cell communication. Their activity is nor- kDa transmembrane glycoprotein (p185HER- mally firmly controlled and regulated. 2/neu) that has high homology with EGFR. Disturbances in the PTK signaling resulting HER-2/neu is overexpressed in about 30% of from mutations and other genetic alterations NSCLCs, particularly in adenocarcinomas contribute to the malignant transformation. and is associated with multiple drug resist- A number of growth factors and their recep- ance phenotype and high prevalence of tors are expressed by lung cancer cells or metastases.3 A point mutation resulting in their neighboring stromal cells, thus produc- the substitution of the amino acid residue 664 ing autocrine or paracrine growth stimulation from valine to glutamic acid is commonly loops. Several are encoded for by proto-onco- found, and this mutation contributes to the genes which become activated in the course malignant transform of affected cells. of the lung cancer development.3 The overex- Alterations and amplifications of HER-2/neu pression of cell cycle regulatory proteins such gene have been reported in NSCLC.20 as cyclin D1,17 cyclin E,18 and cyclin B1,19 en- Chemotherapy combined with trastuzumab hance the cell proliferation, decrease the cel- (Herceptin), a monoclonal antibody against lular apoptotic potential and are commonly the HER2/neu receptor is now under clinical found in NSCLC tumor specimen. trials.3 Epidermal growth factor receptor (EGFR), MYC proto-oncogene belongs to a family also called ErbB-1, is the member of a sub- of related genes (c-MYC, N-MYC, L-MYC) family of closely related proteins. After lig- which encode transcription factors that acti- and-binding, the intracellular tyrosine kinase vate genes involved in the growth control and domain of the EGFR receptor is activated and apoptosis. The MYC phosphoproteins are lo- undertakes autophosphorylation, which initi- calized in the nucleus.22 The transcriptional ates a cascade of intracellular events. A regulation by MYC proteins is mediated by downstream signaling pathway involves the heterodimerizing with partner proteins such activation of p21-Ras and mitogen-activated as MAX, MAD or MX11.23 MYC-MAX het- protein kinases (MAPKs). EGFR signaling is erodimer binds to specific DNA sequences critical for the normal cell proliferation, but named E-box elements in the neighborhood its deregulation is crucial for cancer patho- of promoters of downstream target genes and genesis, neoangiogenesis, metastasis, and activate their transcription. Histone acetylase Radiol Oncol 2005; 39(3): 197-210. 200 Panov SZ / Molecular biology of the lung cancer is activated and leads to alterations in chro- the plasma membrane, where they can effec- matin structure, which, in turn, modulate the tively interact with their upstream activators gene transcription. On the other hand, the and downstream targets. In active state RAS MYC-MAX complex represses a transcrip- proteins binds to guanosine triphosphate tional activation. MAX can bind MAD and (GTP) and through the intrinsic GTPase ac- MX11 proteins to repress transcription, an- tivity and conformational change of RAS, the tagonize MYC, and promote cellular differen- GTP hydrolyze to guanine diphosphate tiation.20 The molecular abnormalities involv- (GDP) and after interacting with its substrate ing the MYC genes or their transcriptional Raf1, RAS returns to the inactive state. The deregulation were found to be an important cell proliferation signal is subsequently trans- molecular mechanism in the pathogenesis of mitted by a cascade of RAS-dependent kinas- human lung cancers.23 The most frequent ab- es, activating the MAPK, which translocate to normality involving MYC members in lung the nucleus and initiate transcription cancer is gene amplification or gene overex- factors.20 This signal transduction pathway is pression without amplification. The overex- sometimes called SOS-Ras-Raf-MAPK mito- pression of a MYC gene, with or without am- genic cascade.11 In malignant cells, the point plification, occurs in 80 to 90% of SCLCs.22 mutation in the RAS gene can make the RAS In contrast to SCLC, the amplification of the protein defective in the intrinsic GTPase ac- MYC gene occurs only in approximately 10% tivity that becomes locked into the growth of NSCLC samples. However, MYC overex- stimulatory GTP-bound form, constantly pression without MYC gene amplification oc- sending the signal stimulating cell prolifera- curs in over 50% of NSCLC investigated spec- tion signals to the nucleus.25 RAS mutations imens.22 MYC gene overexpression has been are very rare or absent in SCLC, but can be identified to be a late event in lung cancer identified in 15-20% of NSCLC. Up to 50% of pathogenesis in the vast majority of SCLCs.20 the lung adenocarcinomas carry RAS muta- Lung tumor cell lines established from tions,26 usually affecting codon 12 of KRAS metastatic tumors have a high frequency of (85% of cases), and rarely codon 13 of HRAS, MYC amplification, and this probably ex- or codon 61 of NRAS gene.23 The majority (up plains the correlation of MYC amplification to 70%) of these mutations are G→T trans- with a poor clinical prognosis.24 The anti- versions that are induced by benzopyrene di- sense oligonucleotides therapy models direct- ethyloxide (BPDE), nitrosamines and other ed at downregulating MYC expression show DNA adducts-forming agents that are present encouraging results in cell culture.3 in the tobacco smoke. It is believed that this The dominant RAS proto-oncogene is ex- is the reason for the correlation between tremely important for the transduction of the smoking history and the frequency of KRAS growth-promoting signals from the mem- mutations in NSCLC samples which are asso- brane to the nucleus and consequently for the ciated with poor prognosis.27 Few clinical tri- cellular proliferation. The RAS family of als are conducted: using vaccination with mu- genes includes: the HRAS gene (homologous tant KRAS peptides, by suppression of the to the oncogene of the Harvey rat sarcoma mutant RAS gene using antisense oligonu- virus), the KRAS2 gene (homologous to the cleotides, or by inhibition of the farnesylation oncogene of the Kirsten rat sarcoma virus) of the RAS protein that is necessary for its ac- and the NRAS gene (initially cloned from hu- tivation.3 man neuroblastoma cells). The RAS genes The distinguishing feature of SCLC tumors code for four highly homologous 21 kDa pro- is the production and release of a broad range teins called p21 anchored to the inner side of of neuropeptides from the neoplastic cells. Radiol Oncol 2005; 39(3): 197-210. Panov SZ / Molecular biology of the lung cancer 201 Angiotensin, bombesin, insulin-like growth ty of histopathological preneoplastic/preinva- factor 1, vasopressin, serotonin, and sub- sive grades. There are a number of other can- stance P are among the best studied signal didate tumor suppressor genes located at 3p molecules released by SCLC cells.28 These and their allelic loss may probably be the ear- peptides act as ligands for high-affinity re- liest acquired genetic abnormality in the lung ceptors on the tumor cell surface, and their cancer pathogenesis.3,30 binding consequently activate the G-protein FHIT is a tumor-suppressor gene located at coupled receptors enabling a further intracel- 3p14.2, coding for a dinucleoside 5’, 5’’’-P1- lular transmission of the proliferative signal. P3-triphosphate hydrolase protein product By this, SCLC cells are self-stimulating the (often denoted as pFHIT). The loss of the growth by autocrine and paracrine manner. gene results in the accumulation of diadeno- sine tetraphosphate, thus stimulating DNA synthesis and cell proliferation. A decreased Insensitivity to anti-growth signals: expression of FHIT has been found in 49% of tumor suppressor genes NSCLC specimen by immunochemistry. pFHIT expression is significantly reduced in a Tumor suppressor genes (TSG) play a critical large number of early-stage NSCLC and pre- role in cell’s antiproliferative circuitry and are neoplastic lesions in chronic smokers. The as- also involved in the cellular response to DNA sociation between cigarette smoking and damage and consequent reparation process- pFHIT expression suggests a role for FHIT in es. There is a frequent loss of tumor suppres- the initiation of smoking-related lung car- sor genes during the pathogenesis and pro- cinogenesis.20 It was demonstrated that the gression of lung cancers, as in many epithe- reintroduction of wild-type FHIT inhibits lung lial cancers. The inactivation of the tumor cancer in vitro growth and in vivo tumori- suppressor genes occurs by loss of one allele genicity in nude (athymic) mice.23 from the chromosomal locus, termed loss of The RARβ. (retinoic acid receptor beta) heterozygosity (LOH) and damage to the oth- gene, located at 3p24 is a strong TSG candi- er allele by gene mutation or the epigenetic date. Low or absent RARβ. expression was hypermethylation of its promoter. The chro- detected with high frequency in lung cancer mosomal regions that where found to be most cell lines and primary lung tumours.23 It ap- frequently affected by LOH in lung carcino- pears to result from the aberrant promoter mas are 1p, 3p, 4p, 4q, 5q, 8p, 9p (p16 TSG lo- methylation of the RARβ and was observed in cus), 9q, 10p, 10q, 13q (RB-retinoblastoma lo- approximately 40% of primary SCLCs. cus), 15q, 17p (p53 locus), 18q, 19p, Xp, and The TP53 tumor-suppressor gene (p53) is Xq.3 The allelic loss at several loci on the located at chromosome arm 17p13.1 and en- chromosome arm 3p is one of the most fre- codes a 53 kDa nuclear protein that acts as a quent and earliest genetic events in lung can- DNA-binding, sequence-specific transcrip- cer pathogenesis found in up to 96% of carci- tion factor that activates the expression of nomas and 78% of preneoplastic bronchoep- genes engaged in promoting growth arrest in ithelial lesions.29 The high frequencies of the G1 phase or cell death in response to the LOH and frequent homozygous deletions genotoxic stress.31 Thus, p53 has a role of found in many lung cancer cell lines and tu- “guardian of the genome”, maintaining the mor samples suggest that few potential tumor genome integrity during the cellular stress suppressor genes reside at this chromosome from DNA damage, hypoxia, and activated region.23 Moreover, the frequency and size of oncogenes. Also, p53 prevents cells with the allelic loss of 3p correlate with the severi- damaged DNA from undergoing mitosis Radiol Oncol 2005; 39(3): 197-210. 202 Panov SZ / Molecular biology of the lung cancer when they enter the G2 phase. p53 blocks tween the cell differentiation and prolifera- cells at the G2 checkpoint, at least partially, tion. The phosphorylation status of the RB by inhibition of cdc2, the cyclin-dependent protein and its interaction with transcription kinase required to enter mitosis. The ability factor E2F is most important for the regula- of p53 to inhibit cellular proliferation or to in- tion of G0/G1 cell cycle transition. When RB duce apoptosis is suppressed by HDM2 pro- is dephosphorylated, it suppresses the G1 to tein product, the human homologue of the S phase transition.32 During G1 phase, cyclin murine double minute 2 (MDM2). This pro- D1 is associated with cyclin-dependent–ki- tein blocks p53 regulation of target genes and nases CDK2 and CDK4 that results in phos- enhances its proteasome dependent degrada- phorylation and activation of RB. Hypo- tion.31 On the other hand, p53 upregulates phosphorylated RB binds the E2F transcrip- the expression of HDM2 by directly binding tion factor, thus blocking the transcription of and activating the HDM2 promoter and thus genes regulating the cell cycle. On the con- p53 is downregulating its own expression. trary, when RB is phosphorylated, E2F disso- This autoregulatory loop keeps p53 at virtual- ciates and activates the transcription, thus fa- ly undetectable levels in normal cells.3 cilitating S phase entry.23 Abnormalities of Missense mutations (mainly G→T transver- the RB gene in lung cancer include deletions, sions) clustered in the middle of the gene at nonsense mutations, pathogenic splicing codons 157, 245, 248, and 273 abolishes its variations and chromosomal deletions. The tumor suppressing activity and extend the disruption of the pRb pathway releases E2Fs p53 mutant protein half-life that can be easi- allowing cell proliferation to proceed and ly detected by immunohistochemistry. The making the cell insensitive to antigrowth fac- p53 gene mutations in lung cancer have been tors that normally function to control a tran- extensively investigated and were found that sition through the G1 phase of the cell cy- p53 is inactivated in 75% of SCLCs and about cle.11 More than 90% of the SCLC and 15-30% 50% of NSCLCs and the frequency of muta- of the NSCLC neoplasms have abnormal or tions correlate with cigarette smoking.20 It is no RB expression.22 Although RB plays an im- intriguing that the mutations at codon 157 ap- portant role in pulmonary cancer pathogene- pear to be unique to pulmonary carcinomas, sis, pRB status has no prognostic significance while codon 248 and 273 hot spots mutations in NSCLC patients.20 occur in other cancers, e.g., colon, liver, and PTEN (Phosphatase Tensin Homolog prostate.22 Nonsmokers who develop lung Deleted on Chromosome Ten) gene is located cancer have a completely different, almost at chromosome 10q23 encodes a lipid phos- random grouping of p53 mutations.22 phatase which dephosphorylates PIP3 and Although the prognostic role of p53 muta- posses tumor suppressor activity in vitro and tions in NSCLC p53 is still under debate, in vivo. Mutations or deletions of the PTEN their presence influences the clinical re- gene have been found in a few lung cancer sponse to cisplatin-based chemotherapy and cell lines and tumor samples.23 radiotherapy.3 Transforming growth factor-β (TGF-β) is The RB tumor-suppressor gene is located multifunctional protein that inhibits the pro- on chromosome 13q14 and its protein prod- liferation of many epithelial cells through uct is a nuclear phosphoprotein initially iden- binding with a set of cell receptors. It is a tified in childhood retinoblastomas. RB pro- checkpoint inhibitor involved in the cell cy- tein cooperates with p53 in the regulation cle regulation, causing cells to cease prolifer- and control of cell cycle progression, the tran- ation and arrest in G1.22 The reduced levels scriptional level, and the equilibrium be- of TGF-β expression was found in NSCLC Radiol Oncol 2005; 39(3): 197-210. Panov SZ / Molecular biology of the lung cancer 203 samples by immunocytochemical staining mutated and p16 is intact in SCLC, while p16 studies. expression is disrupted and Rb is usually in- Another candidate TSG on chromosome tact in NSCLC.22 p19ARF binds to the MDM2- 10q25-26 is DMBT1. It is frequently down p53 and prevents p53 degradation. The loss regulated and occasionally homozygously of p19ARF is more frequent in lung tumours deleted in lung cancer.23 The overexpression with neuroendocrine features.23, 31 or activation of insulin-like growth factor I receptor (IGF-IR) has been observed in many human cancers including pulmonary carcino- Evading apoptosis mas. The p16INK4 (also termed CDKN2A) is a tumor-suppressor gene located on chromo- Apoptosis or programmed cell death is a ge- some 9p21 and codes for two proteins trans- netically controlled process that is essential lated by alternative mRNA splicing: α-tran- for tissue remodeling during embryogenesis script that is translated into p16 (p16INK4) and and for the maintenance of the homeostatic β-transcript that is translated into p14ARF balance of cell numbers during adult life. A protein. p16 protein that is part of the p16-cy- deregulation of cell death pathways is impli- clin D1-Cdk4-RB pathway.32 p16 regulates cated in tumor initiation, progression, and cell-cycle progression through a G1/S restric- drug resistance in many human cancers and is tion point by inhibiting CDK4 and CDK6/cy- one of the hallmarks of cancer.11, 33 Two major clin D-mediated phosphorylation of pRB.20 intracellular apoptosis signaling pathways can The disruption of p16 function results in in- lead to programmed cell death, the mitochon- appropriate hyperphosphorylation and, drial pathway (intrinsic) and the death recep- therefore, inactivation of pRB. The overex- tor (extrinsic) pathway. Mediated by a cascade pression of the E2F transcription factor up- of caspase activations and other mediator pro- regulates p16 expression and inhibits cyclin teins, both pathways finally lead to the prote- D-dependent kinase activity, suggesting the olytic cleavage of a variety of cellular proteins, presence of a feedback loop. p14ARF protein induces DNA fragmentation and numerous binds to and stabilizes HDM2 (MDM2 homo- morphological changes that are characteristic logue), increasing its availability of wild-type of cells undergoing apoptosis. Key genes that p53. The loss of p14ARF or p53, which are regulate apoptosis include the p53 tumour common genetic lesions in lung cancer, per- suppressor gene and the Bcl-2 gene family. mits an amplified MYC free opportunity for Simplified, the BCL-2 family members are ma- the cell proliferation and transformation. jor regulators of the apoptotic process, where- p14ARF appears to bridge a gap between onco- as caspases are the major executioners. genic signals and p53 whereby p14ARF-in- Bcl-2 (B-cell lymphoma-2) gene was the duced activation would be critical to move first oncogene found to function through the the compromised cell toward apoptosis.22, 31 production of an inhibitor of apoptosis. The The expression of p16INK4 gene in NSCLCs is bcl-2 gene family consists of more than 15 frequently altered by abnormal promoter members, which either promote or inhibit the methylation (25% of cases) and homozygous apoptosis.34,35,36 The bcl-2 gene is located on deletions or point mutations (10%-40%).23 It chromosome arm 18q21 and the BCL-2 pro- was found that the disturbances in both, the tein product is localized within the outer mi- p16/pRb and p53 pathways are essential for tochondrial membrane, endoplasmic reticu- the enhanced proliferation of NSCLC cell lum and the nuclear envelope, where it exerts lines. There is an inverse relation between anti-apoptotic effect within many cell types.34 p16 and Rb in pulmonary carcinomas: Rb is Following the apoptotic stimulation, pro- Radiol Oncol 2005; 39(3): 197-210. 204 Panov SZ / Molecular biology of the lung cancer apoptotic proteins are activated through post- sion or degradation of the chromosomes.39 transcriptional modifications or changes in Due to the inability of the conventional DNA their conformation. BCL-2 protein forms het- polymerases to replicate the 5’-end of linear erodimers with proapoptotic BCL-2 family DNA, telomeres shorten during each cell divi- members, leading to their inactivation. In ad- sion in the normal human somatic cells. This dition, BCL-2 proteins may interfere with crit- phenomenon is known as an end-replication ical steps during the integration of proapop- problem. This shortening does not produce totic signals at the level of mitochondria, the loss of the essential genes in which each of thereby abrogating cytochrome-C release. the 46 human chromosomes is capped with BAX is a BCL-2-related protein which pro- long repeats of non-coding DNA sequences motes apoptosis and is a downstream tran- named telomeres. The human telomeres con- scription target of p53. BCL-2 protein het- sists of highly repetitive DNA of tandem se- erodimerizes with BAX consequently inhibits quences TTAGGG)n.40,41 It has been calculat- apoptosis. Tumor cells often escape apoptosis ed that roughly 50–100 bp are lost with each as the normal physiological response when round of cell division.42 Human cells are esti- challenged by cellular and DNA damage. mated to have the potential to undergo on av- BCL-2 overexpression, detected by immuno- erage 50–70 divisions. At this point the cell histochemistry, was found in 75%-95% of growth arrests and enters senescence. A SCLC tumors, 25%-30% of the squamous cell dozen of telomeric proteins are needed to hide carcinomas and in 10% of adenocarcinomas.37 the telomeres from the cellular machinery that The significantly higher incidence of bcl-2 would normally treat the end of a linear DNA overexpression in SCLC is unexpected as molecule as a broken strand needing repair.43 these tumors are more sensitive to chemother- The key telomeric DNA binding proteins are apeutic agents that induce an apoptotic re- the telomeric repeat binding factors, sponse.3 Interestingly, the expression of BAX Tankyrase, heterogeneous nuclear ribonucleo- and BCL-2 proteins is inversely related in neu- proteins and few other functionally related roendocrine cancers. Namely, high BCL-2 and proteins. The physiologic maintenance of the low BAX expression occurs in most SCLC tu- telomere requires complex interactions mors which are also mostly p53 deficient.3 The among these proteins, telomeric DNA, and significance of the bcl-2 expression in lung other cellular factors. Telomere integrity is al- cancer for the overall survival is controversial, so essential for the chromosome numerical but bcl-2 expression was found to be associat- and positional stability and the telomere ed with a better prognosis in NSCLC patients shortening facilitates the evolution of cancer that may be associated with the lower tumor cells by promoting chromosome end-to-end vascularization.20,38 fusions and the development of aneuploidy. The inhibition of telomerase in immortal can- cer-cell lines by genetic or pharmacological Limitless replicative potential - methods results in telomere shortening and telomeres and telomerases eventually halts cell proliferation.44 Telomerase is a specific ribonucleoprotein Telomeres are specialized heterochromatin enzyme complex that elongates and maintains structures at the end of each chromosome that the preexisting telomeres of eukaryotic chro- serves as protective caps and plays a role in mosomes, using an intrinsic RNA molecule as the maintaining chromosome integrity, re- a template and thus is extending the number versibly represses the transcription of neigh- of divisions the cell may undertake.45 boring genes and prevents the end-to-end fu- Telomerase holoenzyme contains two main Radiol Oncol 2005; 39(3): 197-210. Panov SZ / Molecular biology of the lung cancer 205 components that are essential for the activity: hibitors regulating endothelial cell prolifera- hTERT subunit (RNA-directed DNA poly- tion and migration are involved in the process merase, i.e. reverse transcriptase, EC 2.7.7.), of angiogenesis. Growth factors that have and hTR, 451-nt RNA chain that serves as a been shown to stimulate angiogenesis in- template. The enzyme complex also contains clude vascular endothelial growth factor many proteins necessary for the full enzymat- (VEGF), basic fibroblast growth factor ic activity that are collectively named as (bFGF), platelet-derived endothelial cell telomerase-associated proteins. The gene for growth factor (PD-EGF) and platelet-derived the telomerase catalytic subunit hTERT is growth factor (PDGF).3,23 The productions of more than 37 kb in length and consists of 16 angiogenesis factors apparently influence the exons.46 The telomerase activity is absent in clinical outcome of lung cancer patients. the majority of normal cells in adult organ- Namely, the VEGF levels in plasma are corre- isms, but is increased during the development lated with the degree of angiogenesis in and neoplasia.47 Since over 90% of human NSCLC and the VEGF expression was found neoplastic cells have increased telomerase ac- to be associated with the decreased overall tivity, it is now generally accepted that this is and disease-free survival in NSCLC pa- a one of the cancer hallmarks and extremely tients.50 Immunochemical studies demon- frequent and consistent cancer-associated strated that bFGF is a prognostic indicator in molecular abnormality. Generally, the telom- lung adenocarcinoma, since the 5-year sur- erase expression in malignant tumors is deter- vival rate was significantly lower for bFGF mining the capacity for the unlimited prolifer- positive patients and the more aggressive ation and thus immortality. A high telomerase clinical behavior was associated with up-reg- activity was detected in almost 100% of SCLC ulation of PDGF.23 In a few clinical trials, im- and 80% of NSCLC samples using a PCR- pressive results were achieved by targeting based telomeric repeat amplification protocol VEGF with a “humanized” monoclonal anti- (TRAP assay). A high telomerase activity in VEGF antibody. Unfortunately, unexpected primary NSCLC was found to be associated bleeding from large necrotic lung neoplastic with the increased cell proliferation rates and masses occurred in the initial trials, but this advanced pathologic stage.48 Recently, the should be approachable by a more careful pa- telomere shortening was found to be an early tient selection.3 molecular abnormality in bronchioepithelial carcinogenesis, preceding telomerase expres- sion and p53/Rb inactivation that occurs in Tissue invasion and metastasis most high-grade preinvasive lesions.49 Since the telomerase activity is associated with ma- Molecular mechanisms that lead to the com- lignant growth, it is a marker for lung cancer plex ability of the primary lung cancer cells to detection, and a important target for novel invade the adjacent tissue and to disseminate therapeutic approaches.23 to the distant organs of the patient’s body are mainly unknown.3 This process involves degradation of the basement membrane, in- Tumor angiogenesis vasion of the surrounding stroma and the blood or lymphatic vessel, ability to growth New blood vessel growth (neovascularization without adhesion, angiogenesis, cell prolifer- or neoangiogenesis) is required for tumors to ation, and migration.11 Few different genes sustain and grow beyond 3 mm in diameter and their protein products are identified to be and for metastasis. Different inducers and in- important for the process of tissue invasion Radiol Oncol 2005; 39(3): 197-210. 206 Panov SZ / Molecular biology of the lung cancer and metastatic capability of the neoplastic spread of the lung cancer cells. The reduced cells. expression of laminin α chains (α3 and α5) in E-cadherin is a cell adhesion molecule that lung neoplastic tissue might result in the is universally expressed on epithelial cells. basal membrane fragmentation necessary for During the pathogenesis of most epithelial the cancer cell invasion.57 Changes in the in- cancers, E-cadherin function is lost by the tegrin expression are found in metastatic mutational inactivation of the E-cadherin or cells in many human neoplasms, including β-catenin genes, as well as by the transcrip- the lung cancer.11 Recently, a study conduct- tional repression, or enhanced proteolysis. ed by Manda and collaborators, identified This results in reduced E-cadherin-mediated that the LAMB3 gene (coding for the laminin cell-cell adhesion and enables the malignant β3 chain, a component of laminin-5) was ex- cells to invade the tissues and to enter the pressed only in NSCLC cells and not in SCLC blood or lymphatic vessels.51 Therefore, E- tumor cells.58 In the same study, the α6β4 in- cadherin gene is sometimes referred to as the tegrin, the specific laminin-5 binding recep- “suppressor of invasion” gene.52 It was tor, was expressed only in NSCLC cells but demonstrated that E-cadherin loss in lung not in SCLC cells. This suggests that laminin- cancer is associated with the increased metas- 5 might be a critical microenvironmental fac- tasis capability.53 A degradation of the basal tor for the growth of NSCLC tumours.58 membrane and of the extracellular tissue ma- trix by proteases is very important for the lo- cal invasiveness and blood or lymphatic Overview of the molecular abnormalities in metastasis. lung cancer pathogenesis Matrix metalloproteinases (MMPs) are members of the family of zinc-containing pro- The model of lung cancer pathogenesis is de- teolytic enzymes that facilitate the tumor in- picted on the Figure 1 and was developed vasion, the metastatic capabilities, and the tu- based on the previous studies.59 The carcino- mor-related angiogenesis. Conversely, matrix gens from the tobacco or other environmental metalloproteinase inhibitors (MMPIs) have pollutants lead to the loss of the 3p21.3 allele been shown to inhibit tumour growth and in thousands of cells on different sites of the dissemination in preclinical models. It is respiratory epithelium. Later, the tumor sup- therefore not clear why not all lung cancers pressor genes located in the 3p21.3 chromo- express the MMPs and there are conflicting some arm become haplo-insufficient. The reports about the prognostic importance of next hit occurs in genes that are critical for the MMPs expression in lung cancer.54 cell proliferation, such as RB, p53, p16 or oth- It was found that CRMP-1, a protein that er genes either by the mutational inactivation mediates the effect of collapsins, has reduced or by the promoter hypermethylation. That the expression in more aggressive and permits a clonal outgrowth of the initially metastatic lung cancer samples.55 This down- transformed cells. Some authors suggest that regulation is believed to enhance the cell mi- the molecular pathogenesis differs significant- gration ability, which is important for the ly between SCLC and NSCLC main tumor process of metastasis. CRMP and other mem- types.30 It is proposed that during the patho- bers of the collapsin/semaphorin protein genesis of the SCLC neoplastic cells arise di- families might control the cell’s movement.56 rectly either from normal or hyperplastic ep- Laminins and integrins are proteins in- ithelial cells without passing through charac- volved in the adjacent tissue invasion teristic preneoplastic intermediate pathologi- through the basement membrane and further cal stages (parallel theory of lung cancer Radiol Oncol 2005; 39(3): 197-210. Panov SZ / Molecular biology of the lung cancer 207 Figure 1. Main molecular abnormalities occurring during lung cancer pathogenesis (according to References 59 and 22). pathogenesis). On the contrary, the NSCLC tion of the pathogenesis of this complex dis- pathogenesis is accompanied with sequential ease and would led to the advance of novel morphological changes (sequential theory). molecular approaches for the early diagnosis and therapy of the pulmonary carcinomas. Conclusions References Recent progress into the elucidation of the mo- 1. Jemal A, Tiwari RC, Murray T, Ghafoor A, lecular genetic abnormalities involved in the Samuels A, Ward E, et al; American Cancer lung cancer has been achieved using modern Society. Cancer statistics, 2004. Cancer J Clin 2004; technologies for the mutation detection as well 54(1): 8-29. as for the gene expression quantitation using 2. Williams MD, Sandler AB. The epidemiology of microarray techniques. A precise characteriza- lung cancer. 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